Arabian Journal of Chemistry (2017) 10, S928–S935

King Saud University

Arabian Journal of Chemistry

www.ksu.edu.sawww.sciencedirect.com

ORIGINAL ARTICLE

Development and validation of Ketorolac

Tromethamine in eye drop formulation by

RP-HPLC method

* Corresponding author. Tel./fax: +91 4288 234417.

E-mail address: anands17@rediffmail.com (S. Ananda Thangadurai).

Peer review under responsibility of King Saud University.

Production and hosting by Elsevier

http://dx.doi.org/10.1016/j.arabjc.2012.12.031

1878-5352 ª 2014 Production and hosting by Elsevier B.V. on behalf of King Saud University.

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

G. Sunil, M. Jambulingam, S. Ananda Thangadurai *, D. Kamalakannan,

R. Sundaraganapathy, C. Jothimanivannan

Department of Pharmaceutical Analysis, Swamy Vivekanandha College of Pharmacy, Elayampalayam 637 205, Tamil Nadu, India

Received 22 August 2012; accepted 15 December 2012

Available online 4 January 2013

KEYWORDS

Ketorolac Tromethamine;

RP-HPLC method;

PDA detector

Abstract A simple, precise and accurate method was developed and validated for analysis of Ket-

orolac Tromethamine in eye drop formulation. An isocratic HPLC analysis was performed on

Kromosil C18 column (150 cm · 4.6 mm · 5 lm). The compound was separated with the mixture

of methanol and ammonium dihydrogen phosphate buffer in the ratio of 55:45 V/V, pH 3.0 was

adjusted with O-phosphoric acid as the mobile phase at flow of 1.5 mL min�1. UV detection was

performed at 314 nm using photo diode array detection. The retention time was found to be

6.01 min. The system suitability parameters such as theoretical plate count, tailing and percentage

RSD between six standard injections were within the limit. The method was validated according to

ICH guidelines. Calibrations were linear over the concentration range of 50–150 lg mL�1 as indi-

cated by correlation coefficient (r) of 0.999. The robustness of the method was evaluated by delib-

erately altering the chromatographic conditions. The developed method can be applicable for

routine quantitative analysis.ª 2014 Production and hosting by Elsevier B.V. on behalf of King Saud University. This is an open access

article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

1. Introduction

Non-steroidal anti-Inflammatory drugs (NSAIDs) are used forthe management of pain and inflammation associated with

musculo-skeletal, joint disorders and operative procedures.

The uses of these drugs are prevalent in India due to the high

rates of occurrence of rheumatoid disorders. Many NSAIDshave been marketed, one among them is Ketorolac Trometha-mine. It is chemically, (±)-5-benzoyl-2,3-dihydro-1H-pyrroli-

zine-1-carboxylic acid, 2-amino-2-(hydroxymethyl)-1,3-propanediol (Fig. 1). In India it is marketed under the tradename of Acular LS and Acular PS. When administered as

eye drops it demonstrated analgesic, anti-histaminic, anti-inflammatory and anti-pyretic activity. The mechanism of ac-tion is to inhibit prostaglandin biosynthesis and given system-

ically does not cause pupil constriction.Several studies for the estimation of the Ketorolac Trometh-

amine drug using various techniques have been carried out,some of them being; Simultaneous spectrophotometric

Figure 2 UV absorbance spectra.

Figure 1 Chemical structure of Ketorolac Tromethamine.

Development and validation of Ketorolac Tromethamine in eye drop formulation by RP-HPLC method S929

estimation of Ofloxacin and Ketorolac Tromethamine in tabletdosage forms (Fegade et al., 2009). Formulation and evaluation

of mouth dissolving tablet of Ketorolac Tromethamine for themanagement using super disintegrants (Patil et al., 2009).Formulation and development of enteric coated dosage form

using Ketorolac Tromethamine (Rupesh et al., 2010). Stabilityindicating the HPLC method for the simultaneous determina-tion of Moxifloxacin Hydrochloride and Ketorolac Trometha-mine in pharmaceutical dosage form (Naeem et al., 2011).

Revalidation and analytical evaluation of Ketorolac Trometha-mine by HPTLC using reflectance scanning densitometry (Raoet al., 2011). Preparation and evaluation of Ketorolac Trometh-

amine Gel containing genipin for periodontal diseases (Jeonet al., 2007). Absorption ratio method for the estimation ofMoxifloxacin HCl and Ketorolac Tromethamine in their com-

bined dosage form by UV–Visible spectroscopy (Gandhi et al.,2011). Simultaneous determination of Ketorolac Trometha-mine and Furosemide in intestinal perfusion samples by

validated Reversed Phase High Performance Liquid Chroma-tography (Gupta et al., 2005). Formulation and evaluation ofan ophthalmic delivery system of the Non-steroidal Anti-Inflammatory Drug (NSAID), Ketorolac Tromethamine based

on the concept of pH-triggered in situ gelation (Kugalur et al.,2010). Voltametric behavior of Ketorolac and its determinationin HPLC-EC (Squella et al., 2006).

2. Experimental procedures

2.1. Instruments

Waters 2695 with 22996 PDA detector HPLC systems (Em-

power 2 software), Sartorius Electronic Analytical balance,Crest sonicator and Kromosil C18 column (150 cm ·4.6 mm · 5 l) were used.

2.2. Chemicals and reagents

Free gift sample of Ketorolac Tromethamine was obtainedfrom Spectra Pharma Research Laboratories, Hyderabad. A

pharmaceutical product (ACULAR LS and ACULAR PSeye drops) composition containing the same amount of drugformulations was used in the experiments. HPLC grade meth-

anol and ammonium dihydrogen phosphate were procuredfrom Merck Laboratories, Mumbai. HPLC grade deionizedwater was used throughout the experiment.

2.3. Buffer preparation

Ammonium dihydrogen phosphate (5.75 g) was dissolved in

1000 mL of distilled water and adjusted the pH to 3.0 withO-phosphoric acid. It was filtered through 0.45 lm nylonmembrane filter and degassed. It was used as diluents for thepreparation of sample and standard.

3. Method

3.1. Wavelength detection

Accurately weighed Ketorolac Tromethamine equivalent to100 mg in 100 ml volumetric flask, 100 ml of methanol were

added, sonicated for 5 min and filtered through 0.45 lm nylonmembrane filter. Pipette out 1 mL of the above solution anddilute to 10 mL with methanol in 10 mL volumetric flask and

scanned between 200 and 400 nm by UV spectroscopy (Fig. 2).

3.2. Chromatographic conditions

Chromatographic separation was achieved at 30 �C, the detec-tion was carried at 314 nm at a flow rate of 1.5 mL min�1 andrun time was kept at 20 min. Prior to the injection of drugsolution column was equilibrated for 60 min with the mobile

phase flowing through the system. The injection volume was10 lL for assay level. Blank containing the mobile was injectedto check the solvent interference.

3.3. Standard preparation

Ketorolac Tromethamine (100 mg) was weighed and trans-

ferred into a 100 mL volumetric flask and make up to the vol-ume with diluent. Pipette out 1 mL of the above solution isdiluted 10 mL with the mobile phase to get the final concentra-tion of 100 lg mL�1. A representative chromatogram of the

standard was shown in Fig. 3.

3.4. Sample preparation

ACULAR LS and ACULAR PS ophthalmic sample solutionequivalent to 20 mg of Ketorolac Tromethamine was weighedand transferred to 100 mL volumetric flask. To the above solu-

tion was added 70 mL of the mobile phase, sonicated for about15 min and filtered through 0.45 lm nylon membrane filter.1.25 mL of the filtrate was diluted to 10 mL with the mobile

phase. A representative chromatogram of the sample wasshown in Fig. 4 and Table 1.

Figure 3 Standard chromatogram.

Figure 4 Test chromatogram.

Table 1 Analysis of marketed formulations.

Commercial formulation Ingredients Labeled amount

(mg)

Amount found

(mg) n= 6

Found

in (%)

Acular LS eye drops Ketorolac Tromethamine 0.4 0.43 102

Acular PF eye drops Ketorolac Tromethamine 0.4 0.42 101

S930 G. Sunil et al.

3.5. Evaluation of system suitability

10 lL of standard solution was injected in six duplicate be-fore and after the analysis and the chromatograms were re-corded. System suitability parameters like column efficiency,

plate count and tailing factor were also recorded. The columnefficiency determined was found to be more than 2000 USPplate count, USP Tailing for the same peak is not more than

2.0 and % RSD of six injection of the standard solution isnot more than 2.0% the chromatogram as shown in Fig. 5and Table 2.

4. Analytical method validation (ICH, 2005)

4.1. Specificity

Placebo solution was prepared as per the test solution using

equivalent weight of the placebo in a portion. Placebo solutionwas injected into the HPLC system following the test condi-tions, the chromatogram was recorded and measured theresponses of the peaks were noted for any interference of the

excipient at the retention time of Ketorolac Tromethamine(Fig. 6).

Figure 5 System suitability chromatogram.

Figure 6 Placebo (Specificity) chromatogram.

Table 2 System suitability study.

Injection RT Peak area USP plate count USP tailing factor

1 6.085 2606836 2011 1.28

2 6.049 2698061 2008 1.27

3 6.034 2530365 2176 1.22

4 6.021 2591486 2218 1.21

5 5.983 2572701 2136 1.25

Mean 6.0344 2596031.33 2108 1.24

SD 0.037401 61905.95 – –

% RSD 0.006 0.0238 – –

Development and validation of Ketorolac Tromethamine in eye drop formulation by RP-HPLC method S931

4.2. Precision

Precision was measured in terms of repeatability of applicationand measurement. Repeatability of standard application(system precision) was carried out using six replicates of the

sample injection (100 lg mL�1). Repeatability of sample mea-surement (method precision) was carried out in six differentsample preparations from the same homogenous blend of the

marketed sample (100 lg mL�1). The percentage RSD forrepeatability of standard preparation was 1.16% whereas the

% RSD for repeatability of the sample preparation was0.02%. This shows that the precision of the method is satisfac-

tory as percentage RSD is not more than 2% the chromato-gram as shown in Figs. 7 and 8 and Table 3.

4.3. Linearity

The linearity of Ketorolac Tromethamine was determined bypreparing and injecting a solution with a concentration of about50–150 lg mL�1. The calibration curve indicates the response is

Figure 7 Precision chromatogram.

Figure 8 Intermediate precision chromatogram.

Table 3 Precision study of the method.

S. no Sample area % Assay Amount present (mg)

1 2606386 100.4 0.47

2 2698061 103.9 0.48

3 2530365 97.47 0.43

4 2591486 99.82 0.46

5 257270 99.19 0.45

6 2576739 99.25 0.46

MEAN 99.99 0.45

SD 56171.2

% RSD 1.16

S932 G. Sunil et al.

linear over the concentration range studied for Ketorolac Tro-methamine with a correlation coefficient (r) of 0.999.

4.4. Stability of sample solution

The sample solution was prepared as per the test method, and

analyzed initially at different time intervals by keeping thesolution at room temperature. The percentage response

between the initial and different time intervals shows that thesample solutions were stable for at least 24 h at room temper-

ature (Table 4).

4.5. Robustness

Robustness of the method was determined by analyzing thestandard solution at normal operating conditions by changing

Table 4 Stability study.

S. no Time interval (h) Peak response

1 0 2606836

2 2 2698061

3 6 2530365

4 10 2591486

5 16 2572701

6 24 2576739

Mean 2596031.33

SD 51277.56

% RSD 0.019

Development and validation of Ketorolac Tromethamine in eye drop formulation by RP-HPLC method S933

some operating analytical conditions such as flow rate, col-umn, oven temperature, detection wavelength and the mobile

phase. The conditions with variation and their results wereshown in Table 5. The tailing factor is around unity indicativeof peak symmetry and theoretical plate counts were also above

2000. Hence robustness of the extent of variations applied toanalytical conditions was shown in Fig. 9.

4.6. Accuracy

The percentage recovery experiments were performed by add-ing a known quantity of pure standard drug into the pre-ana-lyzed sample. The solution equivalent to 100 mg of Ketorolac

Tromethamine was accurately weighed into a 100 mL volumet-ric flask. The sample was then spiked with standard at level

Figure 9 Robustnes

Table 5 Robustness studies.

System suitability parameters

(variations)

% RSD of peak

response (n= 3

Change in (%) +5 1.20

Organic phase �5 1.23

Change in pH +3 0.019

�3 0.017

Change in flow +1.5 0.004

�1.5 0.003

Change in temp. +25 0.003

50%, 100% and 150% of test concentration. The resultingspiked sample solutions were assayed in triplicate and the re-sults were compared and expressed as percentage. The mean

percentage recovery of Ketorolac Tromethamine was foundto be in the range between 98.54 and 102.5 which are withinthe acceptance limits as shown in Table 6 and Fig. 10.

4.7. Limit of detection (LOD) and limit of quantification

(LOQ)

The limit of detection (LOD) is the lowest amount of analytein a sample that can be detected, but not necessarily quantified,under the stated experimental conditions. LOD and LOQ were

calculated by using standard deviation and slope values ob-tained from the calibration curve by using the formulaLOD = 3.3 (SD/S) and LOQ = 10 (SD/S). The LOD andLOQ values for Ketorolac Tromethamine were found to be

0.116 lg mL�1 and 0.32 lg mL�1 respectively as shown inFigs. 11 and 12 and Table 7.

5. Results and discussion

A different combination of mobile phases and chromato-graphic conditions were tried and a mobile phase containing

ammonium dihydrogen phosphate and methanol (44:55 V/V),Kromosil C18 (150 cm · 4.6 mm · 5 l) column, 1.5 mL min�1

flow rate, 10 lL injection volume, 30 �C column oven tempera-

ture, 314 nm wavelength and 20 min run time was found to be

s chromatogram.

area

)

Mean tailing

factor (n= 3)

Mean retention time

in min (n= 3)

0.67 6.01

0.86 6.05

1.28 6.06

1.30 6.08

1.22 6.75

1.25 6.85

1.19 5.18

Figure 10 Accuracy chromatogram.

Figure 11 LOD.

Table 6 Accuracy study.

Spike level (%) Amount added in (lg mL�1) Peak area % Recovery % Mean recovery

50 0.12 1347374 103.90 102.5

50 1308694 100.83

50 1336027 102.93

100 0.25 2635598 101.54 99.44

100 2575802 99.32

100 2530358 97.48

150 0.36 3532611 96.23 98.54

150 3858796 99.10

150 3906218 100.31

S934 G. Sunil et al.

suitable for all combinations. These chromatographic condi-tions gave retention time of 6.01 min. Specificity of the methodwas checked by injecting the placebo solution, no peaks were

found at the retention time of Ketorolac Tromethamine. Thestability of the sample solution was evaluated by preparing asample solution as per the proposed method and analyzed ini-

tially and at 1 h intervals up to 24 h by keeping the sample solu-tion at room temperature. The results of the stability studiesshowed that the solution of the drug was found to be stable

for 24 h at room temperature. System precision and methodprecision results showed the % RSD of 1.16 and 0.02, respec-tively. A good linearity relationship indicated by correlation

Figure 12 LOQ.

Table 7 LOD and LOQ.

S. no Concentration

(lg mL�1)

Peak area

1 50 1341056

2 70 1814323

3 100 2582490

4 120 3087955

5 150 3877114

Development and validation of Ketorolac Tromethamine in eye drop formulation by RP-HPLC method S935

coefficient (r) value 0.999 was observed between the concentra-tions of 50–150 lg mL�1 of Ketorolac Tromethamine. Interme-

diate Precision was done by changing the analyst, column, withthe same chromatographic conditions and the obtained resultswere within the limits. The Robustness method was evaluated

by deliberately varying the chromatographic conditions of themethod such as mobile phase methanol content, flow rate, col-umn temperature and wavelength. The parameters like tailing

factor and retention time showed adherence to the limits. Theaccuracy of the method was determined and the percentagerecovery was calculated. The data indicate an average of102.5% recovery of the standard sample.

6. Conclusion

The method developed for Ketorolac Tromethamine wasfound to be a simple process and the procedure does not in-volve any experimental conditions. The validation results indi-cated that the method was specific, accurate, linear, precise,

rugged and robust. The runtime was relatively 20 min whichenabled a rapid quantification of many samples in routineand quality control analysis of ophthalmic formulation.

Acknowledgement

The authors are very much thankful to the management andfaculty of Swamy Vivekanandha College of Pharmacy,Elayampalayam for providing support to carry out this work.

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